Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.

The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a crucial process for various cellular activities. The NPC comprises ~30 nucleoporins and is well characterized in vertebrates and yeast. However, only eight plant nucleoporins have been identified, and little information is available about the complete molecular structure of plant NPCs. In this study, an interactive proteomic approach was used to identify Arabidopsis thaliana nucleoporins. A series of five cycles of interactive proteomic analysis was performed using green fluorescent protein (GFP)-tagged nucleoporins. The identified nucleoporins were then cloned and subcellular localization analyses were performed. We found that the plant NPC contains at least 30 nucleoporins, 22 of which had not been previously annotated. Surprisingly, plant nucleoporins shared a similar domain organization to their vertebrate (human) and yeast (Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins exhibited higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358], Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis failed to identify a vertebrate homolog for this protein. Interestingly, Nup136-GFP showed greater mobility on the nuclear envelope than did other nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant development. These findings provide valuable new information about plant NPC structure and function.

Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.

The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca ²⁺ -dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.

Summary: Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole‐targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H⁺‐ATPase, concanamycin A, and an inhibitor of papain‐type cysteine proteinase, E‐64d, abolished both the light‐dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured‐cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain‐type cysteine proteinase(s) under the acidic pH. The light‐dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP‐fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar‐targeting machinery and the organ‐ and stage‐specific differentiation of endomembrane systems in plants.